WO2012169887A2 - Utilisation de nouveaux marqueurs dans un dosage de diagnostic pour la détermination de la sévérité d'une infection par rsv - Google Patents

Utilisation de nouveaux marqueurs dans un dosage de diagnostic pour la détermination de la sévérité d'une infection par rsv Download PDF

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WO2012169887A2
WO2012169887A2 PCT/NL2012/050394 NL2012050394W WO2012169887A2 WO 2012169887 A2 WO2012169887 A2 WO 2012169887A2 NL 2012050394 W NL2012050394 W NL 2012050394W WO 2012169887 A2 WO2012169887 A2 WO 2012169887A2
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rsv
detection
overexpression
olfm4
mmp
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WO2012169887A3 (fr
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Peter Wilhelmus Maria Hermans
Ronald De Groot
Jacob Gerben FERWERDA
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Radboud Universiteit Nijmegen
Stichting Radboud Universitair Medisch Centrum
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Stichting Katholieke Universiteit
Stichting Radboud Universitair Medisch Centrum
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the field of diagnostics, more particularly diagnostics for viral diseases, especially diseases caused by Respiratory
  • RSV Human respiratory syncytial virus
  • bronchiolitis inflammation of the small airways in the lung
  • pneumonia in children under 1 year of age in the United States.
  • RSV can cause bronchiolitis, leading to severe respiratory illness requiring hospitalization and, rarely, causing death. This is more likely to occur in patients that are immunocompromised or infants born prematurely.
  • Other RSV symptoms common among infants include listlessness, poor or diminished appetite, and fever.
  • Recurrent wheezing and asthma are more common among individuals who suffered severe RSV infection during the first few months of life than among controls; whether RSV infection sets up a process that leads to recurrent wheezing or whether those already predisposed to asthma are more likely to become severely ill with RSV has yet to be determined.
  • the amount of fluid used for the lavage and the pressure with which it is applied is variable as is the amount of biological material that is washed out with the fluid. It would therefore be advantageous if there would be an assay that would be feasible on blood or plasma, since this can be obtained more standardized manner and is a common diagnostic procedure of infectious disease. It has appeared, however, that the concentrations of the various inflammatory mediators are different between mucosal tissue, such as the nasopharyngeal tissue, and the blood (see Bermejo-Martin et al, supra). Also, it has been discussed that mechanical ventilation, as is often applied in children with bronchiolitis, influences the mucosa and plasma levels of chemokines and cytokines (Schultz, C.
  • the invention now relates to a method for the prediction of the severity of a disease developing from an infection with human respiratory syncytial virus (RSV) in a subject, comprising the steps of:
  • step b making a prediction on basis of the amount of overexpression detected in step a).
  • said overexpression is preferably detected by a nucleic acid assay or by an immunoassay.
  • the invention relates to a method, wherein the subject is a child, preferably a child having an age of less than 6 months, more preferably a child having an age of less than three months.
  • the invention relates to a method as described above where the detection is performed on a blood sample of said subject.
  • the detection of overexpression of OLFM4 is performed on a cell component derived from peripheral blood mononuclear cells
  • the detection of overexpression of CD177 is performed on a cell component derived from neutrophils
  • the detection of overexpression of MMP8 is performed on a cell component derived from peripheral blood mononuclear cells
  • the detection of overexpression of MMP9 is performed on a cell component derived from peripheral blood mononuclear cells or from neutrophils
  • the detection of overexpression of PTX3 is performed on a cell component derived from peripheral blood mononuclear cells.
  • the invention in a further embodiment relates to a method according to the invention, wherein a subject is predicted to develop a severe disease state if in a sample of said subject OLFM4, CD177, MMP8, MMP9 or PTX3 or combinations thereof are found to be overexpressed by a factor of 1.5, more preferably by a factor of 2, most preferably by a factor of 3 or more.
  • the cell component which is assayed is a nucleic acid, more preferably mRNA.
  • the assay is a PCR assay.
  • the cell component which is assayed is a peptide in which case the assay is preferably an ELISA assay.
  • the invention comprises a detection kit for performing a method according to any of the preceding claims, comprising means for detection of RSV particles and means for the detection of
  • OLFM4, CD177, MMP8, MMP9 or PTX3 or combinations thereof Preferably in such a kit the means for detection overexpression comprise primers, probes or antibodies.
  • a pair of bidirectional primers for detection of overexpression of OLFM4 comprises the primers 5 - atcaaaacacccctgtcgtc- 3 and 5 - gctgatgttcaccacaccac-3'
  • a pair of bidirectional primers for detection of overexpression of CD177 comprises the primers 5 - gggcaggtgtgtcaggag -3 and 5 - ccaccagggttgatgtgagt -3
  • a pair of bidirectional primers for detection of overexpression of MMP8 comprises the primers 5 - CCAGTTTGACATTTGATGCTATCAC -3 and 5 - CTGAGGATGCCTTCTCCAGAA -3
  • Fig. 1 Relative gene expressions in cell fractions of children with RSV infection during acute infection compared with recovery.
  • OLFM4 PBMC pO.001, CD177 PBMC p ⁇ 0.001,CD177 neutrophils pO.001
  • Fig 2 Relative gene expression in different cell fractions in blood from children with a RSV infection categorized by disease severity.
  • *OLFM4 PBMC mild vs sev p ⁇ 0.001, mod vs sev p 0.002.
  • Fig 3 ratio's of OLFM4 transcription after stimulation
  • Fig 6 Plasma olf4 concentrations after 24 hours stimulation with different stimuli
  • Fig. 7 Gene expression levels of MMP-8 and MMP-9 in granulocytes from children with viral lower respiratory tract infections. Relative gene expression levels of MMP-8 (A) and MMP-9 (B) in granulocytes from children during acute RSV positive (RSV+) and RSV negative (RSV-) viral lower respiratory tract infections and after recovery are given. For RSV positive children, relative gene expression levels of MMP-8 (C) and MMP-9 (D) in mild, moderate and severe disease are given. * p ⁇ 0.05, ** p ⁇ 0.01 Fig. 8: Gene expression levels of MMP-8 and MMP-9 in PBMCs from children with viral lower respiratory tract infections.
  • MMP-8 (A) and MMP-9 (B) in PBMCs from children during acute RSV positive (RSV+) and RSV negative (RSV-) viral lower respiratory tract infections and after recovery are given.
  • RSV positive children relative gene expression levels of MMP-8 (C) and MMP-9 (D) in mild, moderate and severe disease are given. * p ⁇ 0.05, ** p ⁇ 0.01
  • Fig. 9 Stimulation of PBMCs and neutrophils by LPS and RSV in vitro.
  • Fig. 10 Relative gene expression of OLFM4 in children with viral lower respiratory infection during the acute phase of infection compared with recovery.
  • PBMCs P 0,0013
  • granulocytes P 0,0053
  • Fig. 11 Relative gene expression of OLFM4 in children with respiratory infection categorized by disease severity.
  • OLFM4 gene expression levels from severe disease in the group ⁇ 3 months vs. >3 months p 0,0233.
  • Fig. 13 Distribution of mild, moderate and severe disease samples in the groups ⁇ 3months and >3 months of age. For ⁇ 3 months: 14,9%, 40,4% and 44,7% for mild, moderate and severe disease respectively. In the >3 months group de distribution is 5,1%, 74,4% and 20,5%.
  • Plasma as used in the present application is used in the normal meaning of the word, i.e. the liquid component of the blood. However, for assaying plasma levels, the assays may be performed on (full) blood, blood serum or plasma.
  • RSV infection is defined as resulting in a "severe disease" state if the patient requires hospitalization, where supportive intervention can be given.
  • This supportive intervention can be mechanical ventilation, treatment with antiviral medicine, and measures to prevent secondary clinical effects and co- infections with other pathogens affecting the respiratory pathways.
  • Children without hypoxia or severe feeding problems are considered to be only mildly affected, those requiring hospitalization for supplemental oxygen (oxygen saturations ⁇ 93%) and/or nasogastric feeding are considered to be moderately affected and children requiring mechanical ventilation are considered to be severely affected (see: Gern J.E. et al., 2002, Pediatr. Allergy Immunol. 13:386- 393; and Wan et al., 1992, Am. Rev. Respir. Dis. 145: 106-109).
  • nucleic acid sequence capable of specific base-pairing with a complementary nucleic acid sequence and binding thereto to form a nucleic acid duplex.
  • a “complement” or “complementary sequence” is a sequence of nucleotides which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson-Crick base-paring rules. For example, the complementary base sequence for 5'-AAGGCT-3' is 3'-TTCCGA-5'.
  • stringent hybridization conditions refers to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize non-specific binding of the primer or the probe to its target nucleic acid sequence.
  • the terms as used includes reference to conditions under which a probe or primer will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background).
  • Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60°C for long probes or primers (e.g. greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary low stringent conditions or "conditions of reduced stringency" include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 2x SSC at 40°C.
  • Exemplary high stringency conditions include hybridization in 50%
  • primer refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase, and at a suitable temperature and pH.
  • the (amplification) primer is preferably single stranded for maximum efficiency in amplification.
  • the primer is an oligodeoxy ribonucleotide.
  • primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization.
  • the exact lengths of the primers will depend on many factors, including temperature and source of primer.
  • a "pair of bi-directional primers" as used herein refers to one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.
  • probe refers to a single-stranded oligonucleotide sequence that will recognize and form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative. Such a probe will often be labelled to enable easy recognition.
  • “Expression” refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) and, if applicable, subsequent translation into a protein.
  • Polynucleotides have "homologous" sequences if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence as described herein. Sequence comparison between two or more polynucleotides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity.
  • the comparison window is generally from about 20 to 200 contiguous nucleotides.
  • the "percentage of sequence homology" for polynucleotides may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and (c) multiplying the result by 100 to yield the percentage of sequence homology.
  • Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms, or by inspection. Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. 1990. J. Mol. Biol.
  • substantially complementary means that two nucleic acid sequences have at least about 65%, preferably about 70%, more preferably about 80%, even more preferably 90%, and most preferably about 98%, sequence complementarity to each other. This means that the primers and probes must exhibit sufficient complementarity to their template and target nucleic acid, respectively, to hybridise under stringent conditions.
  • primer sequences as disclosed in this specification need not reflect the exact sequence of the binding region on the template and
  • a substantially complementary primer sequence is one that has sufficient sequence complementarity to the am lification template to result in primer binding and second-strand synthesis.
  • hybrid refers to a double-stranded nucleic acid molecule, or duplex, formed by hydrogen bonding between complementary nucleotides.
  • hybridise or “anneal” refer to the process by which single strands of nucleic acid sequences form double-helical segments through hydrogen bonding between complementary nucleotides.
  • “Overexpression” or “upregulation” of a gene in a particular cell or sample means that more mRNA is transcribed from the gene in a particular cell or sample than in control cells or samples.
  • said increase in expression can be measured from the concentration of the gene product (protein) in a cell.
  • the increase in expression should amount to at least 1.5 times the expression in controls, preferably at least 2 times, more preferably at least 3 times or more. This increase in expression can be measured against the expression of that gene in a control sample, or, within the same sample against the expression of a control gene.
  • Underexpression or “downregulation” of a gene in a particular cell or sample means that less mRNA is transcribed from the gene in a particular cell or sample than in control cells or samples. Alternatively, said decrease in expression can be measured from the concentration of the gene product
  • the decrease in expression should amount to at least 0.5 times the expression in controls, preferably at least 0.25 times, more preferably at least 0.1 times or less. This decrease in expression can be measured against the expression of that gene in a control sample, or, within the same sample against the expression of a control gene.
  • Thl cytokines like IL-16, 11-2, IL-12p70, IFNy, TNFct
  • Th2 cytokines like IL-13, IL-4, IL-6, 11-10
  • chemokines like IP-10, IL-8, MlPlct (CCL3), MIP-16 (CCL4)
  • RANTES growth factors, like FGFb, PDGFbb, GCSF, VEGF, and IL- 1, IL-1RA and IL-17 (see e.g. Bermejo-Martin, supra).
  • tOLFM4 and CD 177 gene expression is upregulated in circulating leukocytes of patients that will develop a severe RSV infection. It has also been found that gene expression of MMP-8 and MMP-9 was upregulated in circulating leukocytes of patients that will develop a severe RSV infection For MMP-8 this also resulted in an increase of the level of the protein in plasma. It was further found that the expression of pentraxin genes, more specifically pentraxin 3 (PTX3), was upregulated in patients that will develop a severe RSV infection. Also here an increase of the PTX3 protein in plasma was found. As such, an assay for the expression of these genes in circulating leukocytes and/or for the level of the protein concentration in plasma can be used to predict the severity of the disease.
  • PTX3 pentraxin 3
  • OLFM4 (olfactomedin 4, also known as GCl, hGC-1 or GW112) is a gene that was originally cloned from human myeloblasts and found to be selectively expressed in inflamed colonic epithelium.
  • the protein encoded is a member of the olfactomedin-related protein family. The exact function of this gene has not yet been determined.
  • CD 177 is an important neutrophil gene that encodes the neutrophil membrane glycoprotein (gp) NBl.
  • gp neutrophil membrane glycoprotein
  • NBl gp has been studied for more than 20 years and during that time several different names have been used to describe this gp, its antigens, and the gene that encodes this molecule.
  • NBl was first described by Lalezari and colleagues while investigating a case of neonatal alloimmune neutropenia (Lalezari P, et al., 1971, J Clin Invest, 50: 1108-1115).
  • NBl Human Neutrophil Antigen-2a
  • HNA-2a Human Neutrophil Antigen-2a
  • gp carrying this antigen was called NBl gp (Bux, J. et al., 1999, Vox Sang. 77:251).
  • Monoclonal antibodies specific for NBl gp have been produced and clustered as CD 177 (Mason, D. et al, 2002, Blood 99:3877-3880).
  • polycythemia vera (Temerinac, S. et al, 2000, Blood, 95:2569-2576).
  • the coding regions ⁇ and PRV-1 differ at only 4 nucleotides that result in amino acid changes and Caruccio, Bettinotti, and colleagues have shown that PRV-1 and NBl are alleles of a single gene which herein is referred to as
  • CD 177 (Caruccio, L. et al, 2004, Transfusion 44: 77-82). Except for the mentioned effects, the clinical importance of under- or overexpression of CD 177 is unkown.
  • MMPs Matrix metalloproteinases
  • MMPs play a role in cellular migration of neutrophils, lymphocytes and other immune cells to the lungs by degrading extracellular matrix, but also have pro- and anti-inflammatory properties.
  • An imbalance in production, activation, or inactivation of MMPs might augment airway inflammation through direct or indirect effects upon signaling pathways that influence migration of leukocytes through the tissues. (Greenlee et al., 2007, )
  • Increased amounts of MMP-8 and MMP-9 have been observed in respiratory samples and blood obtained from adults and children with acute lung injury and pneumonia as well as in chronic lung diseases such as asthma.
  • MMP-9 protein expression is increased in human airway epithelial cell lines infected with RSV. (Yeo et al, 2002, Arch Virol 147, 229-242) In addition, MMP-9 gene expression is increased in the lungs of RSV-infected mice. (Li & Shen, 2007, Chin Med J (Engl ) 120, 5-11)
  • Pentraxin-related protein PTX3 also known as TNF-inducible gene 14 protein (TSG-14) is a protein that in humans is encoded by the PTX3 gene.
  • Pentraxin 3 (ptx3) is a member of the pentraxin superfamily. This super family characterized by cyclic multimeric structure. PTX3 is rapidly produced and released by several cell types, in particular by mononuclear phagocytes, dendritic cells (DCs), fibroblasts and endothelial cells in response to primary inflammatory signals [e.g., toll-like receptor (TLR) engagement, TNFa, IL-16].
  • TLR toll-like receptor
  • PTX3 binds with high affinity to the complement component Clq, the extracellular matrix component TNFa induced protein 6 (TNAIP6; also called TNF-stimulated gene 6, TSG-6) and selected microorganisms, including Aspergillus fumigatus and Pseudomonas aeruginosa.
  • TNFa induced protein 6 TNFa induced protein 6
  • PTX3 activates the classical pathway of complement activation and facilitates pathogen recognition by macrophages and DCs.
  • the RSV infection localizes in the upper respiratory tract of the body and predominantly affects the mucosal tissues that are present there.
  • the immune reaction that is triggered by the virus thus has a strong local, mucosal character of which the local inflammation (bronchiolitis) is one of the most characteristic phenomena. This thus means that most of the components that are involved in the immune reaction (such as the above listed cytokines and chemokines) will be formed and will act locally. It is thus only logical that the local levels of those compounds will be predominantly influenced by the viral infection, while plasma levels may react more slowly or even remain virtually unaltered. It has also been discussed in the scientific literature that there is a difference between mucosal levels and plasma levels of these compounds. Pitrez et al. (Ann.
  • cytokines produced by in vitro PBMCs may not necessarily reflect the concurrent cytokine pattern production at the mucosal surface in the respiratory tract of infants with acute bronchiolitis. Vieira et al. (J. Bras.
  • the increased expression of the genes coding for the above- mentioned proteins can be detected by nucleic acid based detection methods and by protein based detection methods.
  • the cell component used for the assay can thus be nucleic acid, such as RNA, preferably mRNA, or protein.
  • the reagent is typically an antibody against the protein produced by the gene.
  • the reagent is typically a nucleic acid (DNA or RNA) probe or (PCR) primer.
  • test cell component may be detected directly in situ or it may be isolated from other cell components by common methods known to those of skill in the art before contacting with the reagent (see for example, “Current Protocols in Molecular Biology”, Ausubel et al. 1995. 4th edition, John Wiley and Sons; “A Laboratory Guide to RNA: Isolation, analysis, and synthesis”, Krieg (ed.), 1996, Wiley-Liss; "Molecular Cloning: A laboratory manual", J. Sambrook, E.F. Fritsch. 1989. 3 Vols, 2nd edition, Cold Spring Harbor
  • Detection methods include such analyses as Northern blot analysis,
  • RNA may be detected by such methods as NASBA (L. Malek et al, 1994, Meth. Molec. Biol. 28, Ch. 36, Isaac PG, ed, Humana Press, Inc., Totowa, N.J.) or TMA. These include PCR analyses on microfluid array platforms, allowing simultaneous detection of multiple targets in one sample using limited amounts of input material. Nucleic acid probes, primers and antibodies can be detectably labeled, for instance, with a radioisotope, a fluorescent compound, a bioluminescent compound, a
  • chemiluminescent compound a metal chelator, an enzyme or a biologically relevant binding structure such as biotin or digoxygenin.
  • a metal chelator an enzyme or a biologically relevant binding structure such as biotin or digoxygenin.
  • Such arrays comprise oligonucleotides with sequences capable of hybridizing under stringent conditions to the nucleic acid cell component of which the level is to be detected in a method of the present invention.
  • the invention now provides a nucleic acid assay or immuno assay for the prediction of the severity of an RSV infection and targeting either OLFM4, CD 177, MMP8, MMP9, PTX3 or a combination of one or more of these with one or more chosen from the group of IL8, RANTES and detection of CD4 count (see for a discussion on the relevance of IL-8, RANTES and CD4 count the description of co-pending application PCT/NL2010/050765).
  • Such an assay preferably comprises the following steps:
  • nucleic acid and/or protein optionally isolating the nucleic acid and/or protein from the sample or enriching the sample for the presence of the nucleic acid or protein;
  • cl analyse the gene expression profile of said nucleic acid by assaying it with a nucleic acid assay according to the invention.
  • the samples in the above methods are fresh samples.
  • Gene expression analysis is preferably done using a nucleic acid assay or immunoassay.
  • polynucleotide molecules or proteins from a clinically relevant source in this case e.g. a sample from a patient suspected of RSV infection. Presence of RSV infection may be proven by concomitant assaying for the presence of RSV particles, which can also be done in a nucleic acid assay or immunoassay. The skilled person will be capable of performing these assays, since many of them are commercially available (e.g the QuidelTM Quick View RSV test, the
  • RNA or a nucleic acid derived therefrom e.g., cDNA or amplified RNA derived from cDNA that incorporates an RNA polymerase promoter. If the target molecules consist of RNA, it may be total cellular RNA, poly(A)+ messenger RNA (mRNA) or fraction thereof, cytoplasmic mRNA, or RNA transcribed from cDNA (cRNA).
  • RNA is extracted from cells using guanidinium thiocyanate lysis followed by CsCl centrifugation (Chrigwin et al., (1979) Biochem. 18:5294-5299).
  • total RNA is extracted using a silica-gel based column, commercially available examples of which include RNeasy (Qiagen, Valencia, CA, USA) and
  • RNA isolation reagents examples of which include Trizol (Life Technologies, Breda, The Netherlands) and RNAbee (Tel-Test Inc., Friendswood, Texas, USA).
  • total RNA is extracted using automated nucleic acid extraction platforms an example of which is the Nuclisens EasyMAG (BioMerieux, Durham, NC, USA).
  • Poly(A)+ messenger RNA can be selected, e.g. by selection with oligo-dT cellulose or, alternatively, by oligo-dT or hexamer primed reverse transcription of total cellular RNA.
  • the polynucleotide molecules analyzed by the invention comprise cDNA, or PCR products of amplified RNA or cDNA.
  • the practitioner should take a sample from that subject, and after isolation of the RNA the expression of one or more of IL-8, RANTES, OLFM4, CD 177, MMP8, MMP9 and PTX3 be determined.
  • a control gene or element which is not affected by the infection such as a housekeeping gene
  • the mean value of a pool of control genes can be taken. This correction can, for instance, be done by dividing the expression level of each of the tested genes by the expression level of the control gene(s)/element(s).
  • probes specific for polynucleotides of IL-8, RANTES, OLFM4, CD 177, MMP8, MMP9 or PTX3 may be used to detect and quantify the polynucleotide of the gene in biological fluids or tissue samples.
  • any specimen i.e. blood sample, containing a detectable amount of polynucleotide or encoded polypeptide of the IL-8, RANTES, OLFM4, CD 177, MMP8 or PTX3 genes can be used.
  • a detectable amount of polynucleotide or encoded polypeptide of the IL-8, RANTES, OLFM4, CD 177, MMP8 or PTX3 genes can be used.
  • the peripheral blood mononuclear cells (PBMCs) are isolated from the blood sample.
  • PBMCs peripheral blood mononuclear cells
  • neutrophils from the blood sample are taken.
  • MMP9 both PBMCs and neutrophils could be used.
  • IL-8 For preferred methods for detection of IL-8,
  • Nucleic acid can also be analyzed by RNA in situ methods that are known to those of skill in the art such as by in situ hybridization.
  • the subject can be any mammal, preferably the subject is human and even more preferably, the subject is a child of young age, including prematurely born infants.
  • an antibody specific for the IL-8, RANTES, OLFM4, CD 177, MMP8, MMP9 or PTX3 protein may be used to detect and quantify the presence of the polypeptide produced by the gene in the samples.
  • the invention methods can utilize antibodies immunoreactive with polypeptide encoded by the IL-8, RANTES, OLFM4, CD 177, MMP8, MMP9 or PTX3 genes, the predicted amino acid sequences of which are available from public databases, or immunoreactive fragments thereof.
  • OLFM4 sequence information can be found in the EMBL database under accession numbers, CAH71311.2, AAH47740.1, AAI 17330.1, AAQ88930.1 and EAW52052.1, and in the UniProt database under accession no. Q6UX06.
  • the protein has the following sequence:
  • NP_002415 the sequence can be found under NCBI Reference Sequence: NP_002415.1.
  • this sequence is:
  • the sequence can be found under NCBI Reference Sequence: NP_004985.2.
  • this sequence is:
  • HEFGHALGLD HSSVPEALMY PMYRFTEGPP LHKDDVNGIR HLYGPRPEPE PRPPTTTTPQ PTAPPTVCPT GPPTVHPSER PTAGPTGPPS AGPTGPPTAG PSTATTVPLS PVDDACNV I FDAIAEIGNQ LYLFKDGKYW RFSEGRGSRP QGPFLIADKW PALPRKLDSV FEERLSKKLF FFSGRQVWVY TGASVLGPRR LDKLGLGADV AQVTGALRSG RGKMLLFSGR RLWRFDVKAQ MVDPRSASEV DRMFPGVPLD THDVFQYREK AYFCQDRFYW RVSSRSELNQ VDQVGYVTYD ILQCPED
  • PTX3 the sequence can be found under NCBI Reference Sequence: NP_002843.2.
  • this sequence is:
  • An antibody preparation that consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations can be used.
  • Monoclonal antibodies are made against antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al., Nature, 256: 495, 1975). Both monoclonal and polyclonal antibodies are commercially available, e.g.
  • antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab')2, which are capable of binding an epitopic determinant on genes listed in Tables 1 (and 2).
  • Antibody as used herein shall also refer to other protein or non-protein molecules with antigen binding specificity such as miniantibodies,
  • Monoclonal antibodies can be used in the diagnostic methods of the invention, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • the monoclonal antibodies in these immunoassays can be detectably labelled in various ways. Examples of types of immunoassays that can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the
  • RIA radioimmunoassay
  • sandwich immunometric assay
  • Monoclonal and polyclonal antibodies can be bound to many different carriers to be used to detect the presence of the gene products of the genes of Table 1 and 2.
  • carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.
  • the incubation medium usually added with the labelled soluble antibody.
  • the “blockers” are added to assure that non-specific proteins, proteases, or antiheterophilic immunoglobulins to the immunoglobulins present in the experimental sample do not cross-link or destroy the antibodies on the solid phase support, or the radiolabeled indicator antibody, to yield false positive or false negative results.
  • the selection of "blockers” therefore may add substantially to the specificity of the assays described in the present invention.
  • a number of nonrelevant (i. e., nonspecific) antibodies of the same class or subclass (isotype) as those used in the assays e.
  • g., IgGl, IgG2a, IgM, etc. can be used as "blockers".
  • concentration of the "blockers” (normally 1- 100 ⁇ g/ ⁇ L) may be important, in order to maintain the proper sensitivity yet to inhibit any unwanted interference by mutually occurring cross -reactive proteins in the specimen.
  • a detection kit comprising means for the detection of OLFM4, CD 177, MMP8, MMP9 or PTX3 and/or combinations thereof or combinations with IL-8 and RANTES.
  • a detection kit can be used by the pediatrician to predict the severity status of the disease that the child will develop after RSV infection.
  • the detection kit also comprises means to detect viral particles of the virus. Normally, the test for identifying the viral cause of the disease is done separately, but of course it is more efficient to confirm the diagnosis of RSV infection concomitantly with the assay for predicting the severity of the disease that will develop.
  • the infection should be well established and local and systemic immune responses should already have developed.
  • many tests are commercially available, such as the Sure-Vue® test kit of Fisher Healthcare, the NOW RSV assay of Meridian Bioscience, the SimulFluor® RSV/Para 3 assay of Milli ore, and many others.
  • the tests to detect the presence of RSV can be based on immunological techniques, but may also be based on nucleic acid detection, such as (RT-)PCR.
  • NPA nasopharyngeal aspirate
  • 5 ml blood sample identified as 'acute sample'
  • a recovery NPA and blood sample identified as 'recovery sample'
  • the included subjects were subdivided into 3 groups based on severity of disease; mild (no supportive treatment), moderate (supplemental oxygen and/or nasogastric feeding) and severe (mechanical ventilation).
  • NPA 300 ⁇ was taken for virus detection with multiplex PCR as previously described.
  • the PCR had the capacity of simultaneously detection of fifteen viruses: RSV, hMPV, hBoV, Parechovirus, Adenovirus, Rhinovirus, Enterovirus, Parainfluenza 1-4, Influenza A and B and Coronaviruses OC43 and 229E.
  • RSV mono-infections were further analyzed.
  • the acute blood samples were obtained in a sodium heparin tube.
  • Cell differentiation with lymphoprep was performed to separate the cell fractions: PBMCs and neutrophils.
  • the different cell fractions were stored in Trizol until further processing.
  • the recovery samples were handled in the same way. At the end of this process four tubes per child were available for analysis.
  • RNA cleanup protocol with the RNeasy Kit (Qiagen). 1 pg of the total RNA was converted to cDNA using the RT-PCR protocol: first-strand cDNA synthesis using Superscript III (Invitrogen).
  • the region containing the OLFM4 gene was amplified using the forward 5 - atcaaaacacccctgtcgtc- 3 and reverse 5 - gctgatgttcaccacaccac-3 primer.
  • the CD 177 gene was amplified using the forward 5 - gggcaggtgtgtcaggag -3 and reverse 5 - ccaccagggttgatgtgagt -3 primer.
  • the forward primer 5 - cgtcacacttcatgatggagttg-3 and the reverse primer 5 -cttccttcctgggcatgga - 3 were used.
  • the expression of the different genes was measured by quantitative Sybergreen® real time PCR with the ABI 7500 Fast Real-Time PCR system and software. All samples were run for 40 cycles in duplicate, taking the mean of the cycle threshold (Ct) values as measured value.
  • Ct values of OLFM4 en CD 177 were normalized against the reference gene actin. Relative quantities of mRNA were calculated using a (2- Ct) transformation (Livak, K.J. et al., 2001, Methods 25:402-408)
  • the stimuli were RSV-A2, muramyldipeptide (MDP), Pam3Cys (P3C), E.coli endotoxin (LPS), R848, G-CSF and prednisolon and R848.
  • the plates were incubated at 37°C for respectively 4 and 24 hours.
  • a p-value of less than 0.05 was considered statistically significant.
  • the expression of OLFM4 in PBMCs in the acute samples was significantly higher compared to the recovery samples (p ⁇ 0.001), whereas in the neutrophils no significant up regulation was found.
  • the CD 177 transcripts in both the neutrophils and the PBMCs were significantly upregulated in the acute compared with the recovery samples, (pO.001 and pO.001,
  • OLFM4 could be detected by ELISA in the supernatant after 24 hours incubation with different stimuli.
  • concentration of OLFM4 protein was higher after stimulation with RSV, MDP and R848 in comparison with RPMI. Low concentrations were detected after stimulation with G-CSF and prednisolon. In P3C and LPS a higher
  • Another possible influential factor on OLFM4 transcription could be low oxygen levels. Children with a severe infection have lower oxygen levels in comparison with children with mild and moderate infections.
  • OLFM4 plasma concentrations were not significantly associated with RSV-infection or severity. This is likely to be caused by the small sample size. It is submitted that plasma OLFM4 will appear to be significantly upregulated in plasma in severe infections, which will enable the development of a possible rapid test to predict RSV severity.
  • CD 177 is known to be exclusively expressed on neutrophils and is reported to be increased in severe bacterial infections and myeloproliferative disorders (Stroncek, D.F., 2007, Curr. Opin. Hematol.
  • CD 177 expression in neutrophils and PBMCs was significantly upregulated in acute RSV-infection. In both cell types we found significantly higher expression of CD177 in severe compared with mild and moderate infection. In PBMCs there also was a significant upregulation between the moderate and the mild infection. In our study we found CD 177 expression in the PBMC s, at lower base-line levels than the neutrophils, which may be caused by contamination by neutrophils after lymphoprep separation.
  • Viral LRTI was defined as an acute infection of the lower airways, characterized by increased respiratory effort (tachypnea and/or use of accessory respiratory muscles) and/or expiratory wheezing and/or crackles and/or apnea) in combination with a laboratory confirmed viral etiology.
  • Written informed consent was obtained from all parents and the study was approved by the Committee on Research involving Human Subjects of the University Nijmegen Medical Centre.
  • a blood sample and nasopharyngeal aspirate was collected and parents from hospitalized children were asked permission to draw a second blood sample and nasopharyngeal aspirate 4 to 6 weeks after admission.
  • Medical history, demographics and clinical parameters were collected from questionnaires and medical records. Patients were classified into three different groups based on severity of disease. Children without hypoxia or severe feeding problems were allocated in the mild group, those requiring hospitalization for supplemental oxygen (oxygen saturations ⁇ 93%) and/or nasogastric feeding in the moderate group and children requiring mechanical ventilation in the severe group.
  • a nasopharyngeal aspirate was collected by introducing a catheter, connected to a collection tube and an aspiration system, into the
  • nasopharyngeal cavity 1.5 ml of saline was instilled into the catheter and, while slowly retracting the catheter, the nasopharyngeal fluid was aspirated in a collection tube. Afterwards the catheter was flushed with 1 ml of saline and added to the collection fluid. The samples were kept cold and immediately transferred to the laboratory. The nasopharyngeal aspirate was centrifuged at 500 g for 10 minutes at 4°C and the supernatant was frozen at - 80°C.
  • PBMCs were obtained by density gradient centrifugation
  • Plasma samples were stored at -80°C for ELISAs.
  • RNA from PBMC and granulocytes was extracted using Trizol (Invitrogen Life Technologies) according to the manufacturers' protocol.
  • RNA total RNA
  • RNeasy Minikit Qiagen
  • Total RNA (2 ug, measured with spectrophotometry, Nanodrop) was reverse transcribed using a high-capacity cDNA reverse transcription kit according to the manufacturers' instructions (Applied Biosystems, Foster City, CA) and cDNA was stored at -20 degrees.
  • the relative gene expression was measured with SYBR Green PCR Mastermix (Applied Biosystems; P/N 4367659) on the ABI 7500 Fast Real Time PCR system using standard program and software. After 40 repetitions a dissociation curve was performed as control for the specificity of the PCR reaction.
  • the following primers were used: hActin FW:
  • hMMP-9 FW: GCCCCCCTTGCATAAGGA, hMMP-9 RV:
  • nasopharyngeal aspirate and supernatants of cell stimulation assay were measured by ELISA according to the manufacturers' protocol (DuoSet, R&D systems).
  • TIMP-1 concentrations in plasma were determined as described above.
  • PBMCs and granulocytes were obtained by density gradient centrifugation (Lymphoprep®, Axis Shield, Norway). After washing, PBMCs were brought at a concentration of 5 x 10 6 cells/ml in serum free RPMI (Gibco, Invitrogen, Paisley, United Kingdom) with 100 U/ml of penicilin/streptavidin (Gibco, Invitrogen, Paisley, United Kingdom).
  • Granulocytes were purified by lysing the red blood cells (0.155 M NH4CI, 0.0001 M Na 2 EDTA and 0,01 M KHCO3 ⁇ 4 and, after washing, granulocytes were suspended at a concentration of 5 x 10 6 cells/ml in RPMI supplemented with 0.5% Human Serum Albumin (Sanquin, Amsterdam, The Netherlands)
  • Mononuclear cells (5 x 10 5 in 100 ⁇ ) were added to round-bottom 96- well plates and stimulated with either 100 ⁇ culture medium (negative control), lng/ml LPS (Escherichia coli serotype 055: B5, Sigma Aldrich, purified as described previously (Hirschfeld et at., 2000) or MOI 1 of RSV A2 (kindly provided by Dr. R. de Swart, Erasmus MC, Rotterdam, The
  • RSV A2 was cultured in HeLa cells and purified by ultra- centrifuge over a sucrose 30% gradient. After incubation for 24 hours at 37 °C and 5% CO2. supernatant was collected and stored at -80 °C. Neutrophils (5 x 10 5 in 100 ⁇ ) were stimulated and incubated in the same way for 4 hours and supernatant was stored at-80 °C. Apoptosis was determined on the FACScalibur by Annexin V apoptosis detection kit (BD) according to the manufacturers' instructions and no differences between stimuli were founds after 4 hours.
  • BD Annexin V apoptosis detection kit
  • Values are expressed as percentages for categorical variables and as mean and standard error (SE) or median and interquartile range (IQR) for continuous variables.
  • SE standard error
  • IQR median and interquartile range
  • nasopharyngeal fluid were measured compared to those with mild disease (Table 5). However, no significant differences in MMP-9 concentrations in children with severe disease compared to those with mild and moderate disease were observed.
  • MMP-8, MMP- 9 and TIMP-1 plasma levels were observed between RSV positive and RSV negative children during acute viral respiratory infection (Table 4).
  • MMP-8 and MMP-9 concentrations were higher in nasopharyngeal samples compared to plasma.
  • MMP-9 plasma concentrations are correlated with the number of granulocytes
  • MMP-8 and MMP-9 mRNA and protein expression by PBMCs and neutrophils is not induced by RSV in vitro
  • TIMP-1 is an inhibitor of the protease activity of all known MMPs.
  • MMP-9 is an enzyme that catalyzes the hydrolysis of a protein
  • TIMP-1 is an enzyme that catalyzes the hydrolysis of a protein
  • tissue degradation and airflow obstruction in asthma and chronic bronchitis.
  • tautino et al. 1999, J Allergy Clin, Immunol 104, 530-533;
  • nasopharyngeal samples were increased during acute infection compared to recovery samples no association with disease severity was observed.
  • the wide range of nasopharyngeal concentrations between individuals or the fact that upper respiratory samples do not necessarily represent the situation in the lower airways may explain this observation.
  • MMP-8 and MMP-9 mRNA and protein expression in PBMCs and granulocytes were not induced by stimulation with RSV.
  • Other factors than direct interaction between RSV and host cells could explain the increased gene expression levels of MMP-8 and MMP-9 in children with viral LRTI.
  • Influx of bone marrow-derived neutrophil precursors in blood from children with severe RSV infections can result in higher MMP-9 expression due to granule protein production, such as MMP-8 and MMP-9, during immature stages of neutrophil development.
  • Viral LRI acute viral lower respiratory infection
  • nasopharyngeal aspirate for virology
  • medical history for virology
  • demographics for virology
  • clinical parameters were obtained in the acute stage of the disease. They were asked to return 4-6 weeks later for a recovery sample.
  • the included patients were subdivided into three groups based on the severity of the respiratory infection: mild (no supportive treatment), moderate (supplemental oxygen and/or nasogastric feeding) and severe disease for those who needed mechanical ventilation.
  • a nasopharyngeal aspirate was collected by introducing a catheter, connected to a collection tube and an aspiration system, into the nasopharyngeal cavity. Then, 0.5 ml of sa line was instilled into the catheter and, while slowly retracting the catheter, the nasopharyngeal fluid was aspirated in a collection tube. Afterwards the catheter was flushed with 1 ml of saline and added to the collection fluid. The samples were kept cold and immediately transferred to the laboratory. The nasopharyngeal aspirates were processed according to the protocol (see appendix).
  • RNA from PBMCs and granulocytes was extracted using Trizol (Invitrogen Life Technologies) according to the manufacturers' protocol. RNA cleanup was performed with the RNeasy Minikit (Qiagen). See also the protocols in the appendix. Total RNA was measured with the Nanodrop spectrophotometer.
  • RNA was reversed transcribed into cDNA using Superscript II I (invitrogen). When the amount of RNA in a sample was very low, 22 ⁇ of the sample was used. Large amounts of total RNA were diluted with dH20 to get a solution of 2 ⁇ g RNA in 22 ⁇ . cDNA was stored at -20°C.
  • hActin FW CGTCACACTTCATGATGGAGTTG
  • hActin RV CGTCACACTTCATGATGGAGTTG
  • OLFM4 affymetrix, whole blood children, RSV and/or homo sapiens. More than 90 microarray studies were found. 18 studies were selected based on the population (children/infants), sample size and type of the disease (viral/bacterial, infection/ auto-immune disease, lung diseases). From the series matrix files the results on OLFM4 gene expression were selected and analyzed to look for gene expression up or down regulation to gain insight in regulatory pathways for OLFM4 and specificity in viral infections. All data were log transformed and statistically analyzed.
  • the Chi square test and the Kruskal Wallis test were performed to analyze patient characteristics in the different groups.
  • the Mann- Whitney U-test the unpaired T-test and the Wilcoxon matched-pairs signed rank test were used.
  • the microarray data were analyzed with the paired a nd unpaired T-test (normal distributed data) or the Mann-Whitney U test for not normally distributed data (tested with the Shapiro Wilk Normality test). A value of P ⁇ 0.05 is considered to be statistically significant.
  • OLFM4 gene expression is a marker for infection
  • OLFM4 a marker for viral lower respiratory infection in children.
  • Co morbidity asthma, eczema, bronchopulmonary dysplasia, congenital lung disease, congenital heart disease, milk allergy, immunodeficiency.
  • OLFM4 gene expression is a marker for viral lower respiratory infection because both in PBMCs as well as in granulocytes OLFM4 is significantly higher in the acute samples compared to the recovery samples. Also statistical differences were found between moderate and severe disease in the PBMC samples and between moderate and severe and moderate versus mild disease in granulocytes. This makes OLFM4 gene expression a good biomarker for disease severity.

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Abstract

L'invention concerne une méthode de prédiction de la sévérité d'une maladie se développant à partir d'une infection par le virus respiratoire syncytial (RSV) humain chez un sujet, comprenant la détection de la surexpression de OLFM4, CD177, MMP8, MMP9, PTX3 ou d'une combinaison de l'un ou plusieurs de ceux-ci avec un ou plusieurs éléments choisis dans le groupe de IL8, RANTES et compte de CD4. La surexpression peut être détectée dans un échantillon sanguin par un dosage d'acide nucléique ou par l'intermédiaire d'un immuno-dosage.
PCT/NL2012/050394 2011-06-06 2012-06-06 Utilisation de nouveaux marqueurs dans un dosage de diagnostic pour la détermination de la sévérité d'une infection par rsv Ceased WO2012169887A2 (fr)

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WO2018071498A1 (fr) * 2016-10-11 2018-04-19 Yale University Méthodes de détection d'infection virale respiratoire
WO2019072789A1 (fr) * 2017-10-09 2019-04-18 Medizinische Hochschule Hannover Diagnostic et thérapie pour le virus syncytial respiratoire humain (hrsv)
CN113174434A (zh) * 2021-04-26 2021-07-27 中山大学孙逸仙纪念医院 成人社区获得性肺炎早期检测试剂盒及其应用
JP2021170009A (ja) * 2020-04-16 2021-10-28 国立研究開発法人国立国際医療研究センター 呼吸器感染症の重症化の予測を補助する方法、バイオマーカーの測定値をモニタリングする方法、これらの方法に用いられる試薬キット、呼吸器感染症の重症化の予測を補助する装置およびコンピュータプログラム
WO2022122959A1 (fr) * 2020-12-10 2022-06-16 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation de cd177 comme biomarqueur d'aggravation chez les patients atteints de covid-19
CN118048453A (zh) * 2022-11-29 2024-05-17 广东医科大学附属医院 嗅素结构域家族蛋白4（olfm4）在结肠癌检测产品中的应用
US12110564B2 (en) 2018-05-07 2024-10-08 Yale University Test to distinguish viral-only from bacterial infection or viral/bacterial coinfection using a respiratory swab

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